The present invention is in the field of xcex1-O-linked glycopeptides. In particular, the present invention relates to methods for the preparation of xcex1-O-linked glycoconjugates with clustered glycodomains which are useful as anticancer therapeutics. The present invention also provides novel compositions comprising such xcex1-O-linked glycoconjugates and methods for the treatment of cancer using these glycoconjugates.
Throughout this application, various publications are referred to, each of which is hereby incorporated by reference in its entirety into this application to more fully describe the state of the art to which the invention pertains.
The role of carbohydrates as signaling molecules in the context of biological processes has recently gained prominence. M. L. Phillips, et al., Science, 1990, 250, 1130; M. J. Polley, et al., Proc. Natl. Acad. Sci. USA, 1991 88, 6224: T. Taki, et al., J. Biol. Chem., 1996, 261, 3075; Y. Hirabayashi, A. Hyogo, T. Nakao, K. Tsuchiya, Y. Suzuki, M. Matsumoto, K. Kon, S. Ando, ibid., 1990, 265, 8144; O. Hindsgaul, T. Norberg, J. Le Pendu, R. U. Lemieux, Carbohydr. Res. 1982, 109, 109; U. Spohr, R. U. Lemieux, ibid., 1988, 174, 211). The elucidation of the scope of carbohydrate involvement in mediating cellular interaction is an important area of inquiry in contemporary biomedical research. The carbohydrate molecules, carrying detailed structural information, tend to exist as glycoconjugates (cf. glycoproteins and glycolipids) rather than as free entities. Given the complexities often associated with isolating the conjugates in homogeneous form and the difficulties in retrieving intact carbohydrates from these naturally occurring conjugates, the applicability of synthetic approaches is apparent. (For recent reviews of glycosylation see: Paulsen, H.; Angew. Chemie Int. Ed. Engl. 1982, 21, 155; Schmidt, R. R., Angew. Chemie Int. Ed. Engl. 1986, 25, 212; Schmidt, R. R., Comprehensive Organic Synthesis,Vol. 6, Chapter 1(2), Pergamon Press, Oxford, 1991; Schmidt, R. R., Carbohydrates, Synthetic Methods and Applications in Medicinal Chemistry,Part I, Chapter 4, VCH Publishers, Weinheim, New York, 1992. For the use of glycals as glycosyl donors in glycoside synthesis, see Lemieux, R. U., Can. J. Chem., 1964, 42, 1417; Lemieux, R. U., Fraiser-Reid, B., Can. J. Chem. 1965, 43, 1460; Lemieux, R. U.; Morgan, A. R., Can. J. Chem. 1965, 43, 2190; Thiem, J., et al., Synthesis 1978, 696; Thiem, J. Ossowski, P., Carbohydr. Chem., 1984, 3, 287; Thiem, J., et al., Liebigs Ann. Chem., 1986, 1044; Thiem, J. in Trends in Synthetic Carbohydrate Chemistry, Horton, D., et al., eds., ACS Symposium Series No. 386, American Chemical Society, Washington, D.C., 1989, Chapter 8.)
The carbohydrate domains of the blood group substances contained in both glycoproteins and glycolipids are distributed in erythrocytes, epithelial cells and various secretions. The early focus on these systems centered on their central role in determining blood group specificities. R. R. Race; R. Sanger, Blood Groups in Man, 6th ed., Blackwell, Oxford, 1975. However, it is recognized that such determinants are broadly implicated in cell adhesion and binding phenomena. (For example, see M. L. Phillips, et al., Science 1990, 250, 1130.) Moreover, ensembles related to the blood group substances in conjugated form are encountered as markers for the onset of various tumors. K. O. Lloyd, Am. J. Clinical Path., 1987, 87, 129; K. O. Lloyd, Cancer Biol., 1991, 2, 421. Carbohydrate-based tumor antigenic factors have applications at the diagnostic level, as resources in drug delivery or ideally in immunotherapy. Toyokuni, T., et al., J. Am. Chem Soc. 1994, 116, 395; Dranoff, G., et al., Proc. Natl. Acad. Sci. USA 1993, 90, 3539; Tao, M -H.; Levy, R., Nature 1993, 362, 755; Boon, T., Int. J. Cancer 1993, 54, 177; Livingston, P. O., Curr. Opin. Immunol. 1992, 4, 624; Hakomori, S., Annu. Rev. Immunol. 1984, 2, 103; K. Shigeta, et al., J. Biol. Chem. 1987, 262, 1358.
The present invention provides new strategies and protocols for glycopeptide synthesis. The object is to simplify such preparations so that relatively complex domains can be assembled with high stereospecifity. Major advances in glycoconjugate synthesis require the attainment of a high degree of convergence and relief from the burdens associated with the manipulation of blocking groups. Another requirement is that of delivering the carbohydrate determinant with appropriate provision for conjugation to carrier proteins or lipids. Bernstein, M. A.; Hall, L. D., Carbohydr. Res. 1980, 78, Cl; Lemieux, R. U., Chem. Soc. Rev. 1978, 7, 423; R. U. Lemieux, et al., J. Am. Chem. Soc. 1975, 97, 4076. This is a critical condition if the synthetically derived carbohydrates are to be incorporated into carriers suitable for clinical application.
Antigens which are selective (or ideally specific) for cancer cells could prove useful in fostering active immunity. Hakomori, S., Cancer Res., 1985, 45, 2405-2414; Feizi, T., Cancer Surveys 1985, 4, 245-269. Novel carbohydrate patterns are often presented by transformed cells as either cell surface glycoproteins or as membrane-anchored glycolipids. In principle, well chosen synthetic glycoconjugates which stimulate antibody production could confer active immunity against cancers which present equivalent structure types on their cell surfaces. Dennis, J., Oxford Glycostems Glyconews, Second Ed., 1992; Lloyd, K. O., in Specific Immunotherapy of Cancer with Vaccines, 1993, New York Academy of Sciences, pp.50-58. Chances for successful therapy improve with increasing restriction of the antigen to the target cell. For example, one such specific antigen is the glycosphingolipid isolated by Hakomori and collaborators from the breast cancer cell line MCF-7 and immunocharacterized by monoclonal antibody MBrl. Bremer, E. G., et al., J. Biol. Chem. 1984, 259, 14773-14777; Menard, S., et al., Cancer Res. 1983, 43, 1295-1300.
The surge of interest in glycoproteins (M. J. McPherson, et al., eds., PCR A Practical Approach, 1994, Oxford University Press, Oxford, G. M. Blackburn; M. J. Gait, Eds., Nucleic Acids in Chemistry and Biology, 1990, Oxford University Press, Oxford; A. M. Bray; A. G. Jhingran; R. M. Valero; N. J. Maeji, J. Org. Chem. 1944, 59, 2197; G. Jung; A. G. Beck-Sickinger, Angew Chem. Int. Ed. Engl. 1992, 31, 367; M. A. Gallop; R. W. Barrett; W. J. Dower; S. P. A. Fodor; E. M. Gordon, J. Med. Chem. 1994, 37, 1233; H. P. Nestler; P. A. Bartlett; W. C. Still, J. Org. Chem. 1994, 59, 4723; M. Meldal, Curr. Opin. Struct. Biol. 1994, 4, 673) arises from heightened awareness of their importance in diverse biochemical processes including cell growth regulation, binding of pathogens to cells (O. P. Bahl, in Glycoconjugates: Composition, structure, and function,H. J. Allen, E. C. Kisailus, Eds., 1992, Marcel Dekker, Inc., New York, p.1), intercellular communication and metastasis (A. Kobata, Acc. Chem. Res. 1993, 26, 319). Glycoproteins serve as cell differentiation markers and assist in protein folding and transport, possibly by providing protection against proteolysis. G. Opdenakker, et al., FASEB J. 1993, 7, 1330. Improved isolation techniques and structural elucidation methods (A. De; K. -H. Khoo, Curr. Opin. Struct. Biol. 1993, 3, 687) have revealed high levels of microheterogeneity in naturally-produced glycoproteins. R. A. Dwek, et al., Annu. Rev. Biochem. 1993, 62, 65. Single eukaryotic cell lines often produce many glycoforms of any given protein sequence. For instance, erythropoietin (EPO), a clinically useful red blood cell stimulant against anemia, is glycosylated by more than 13 known types of oligosaccharide chains when expressed in Chinese hamster ovary cells (CHO) (Y. C. Lee; R. T. Lee, Eds., Neoglycoconjugates: Preparation and Applications, 1994, Academic Press, London). The efficacy of erythropoietin is heavily dependent on the type and extent of glycosylation (E. Watson, et al., Glycobiology, 1994, 4, 227).
Elucidation of the biological relevance of particular glycoprotein oligosaccharide chains requires access to pure entities, heretofore obtained only by isolation. Glycoprotein heterogeneity renders this process particularly labor-intensive. However, particular cell lines can be selected to produce more homogeneous glycoproteins for structure-activity studies. U.S. Pat. No. 5,272,070. However, the problem of isolation from natural sources remains difficult.
Receptors normally recognize only a small fraction of a given macromolecular glycoconjugate. Consequently, synthesis of smaller but well-defined putative glycopeptide ligands could emerge as competitive with isolation as a source of critical structural information (Y. C. Lee; R. T. Lee, Eds., supra).
Glycoconjugates prepared by total synthesis are known to induce mobilization of humoral responses in the murine immune system. Ragupathi, G., et al., Angew. Chem. Int. Ed. Engl. 1997, 36, 125; Toyokuni, T.; Singhal, A. K., Chem. Soc. Rev. 1995, 24, 231; Angew. Chem. Int. Ed. Engl. 1996, 35, 1381. Glycopeptides, in contrast to most glycolipids and carbohydrates themselves, are known to bind to major histocompatability complex (MHC) molecules and stimulate T cells in favorable cases. Deck, B., et al., J. Immunology 1995, 1074; Haurum, J. S., et al., J. Exp. Med. 1994, 180, 739; Sieling, P. A., et al., Science 1995, 269, 227 (showing T cell recogniztion of CD1-restricted microbial glycolipid). Properly stimulated T cells express receptors that specifically recognize the carbohydrate portion of a glycopeptide. The present invention demonstrates a means of augmenting the immunogenicity of carbohydrates by use of a peptide attachment.
Preparation of chemically homogeneous glycoconjugates, including glycopeptides and glycoproteins, constitutes a challenge of high importance. Bill, R. M.; Flitsch, S. L.; Chem. and Biol. 1996, 3, 145. Extension of established cloning approaches to attain these goals are being actively pursued. Various expression systems (including bacteria, yeast and cell lines) provide approaches toward this end, but, as noted above, produce heterogeneous glycoproteins. Jenkins, N., et al., Nature Biotech. 1996, 14, 975. Chemical synthesis thus represents a preferred avenue to such bi-domainal constructs in homogeneous form. Moreover, synthesis allows for the assembly of constructs in which selected glycoforms are incorporated at any desired position of the peptide chain.
Prior to the subject invention, methods of glycopeptide synthesis pioneered by Kunz and others allowed synthetic access to homogenous target systems both in solution and solid phase (M. Meldal, Curr. Opin. Struct. Biol, 1994, 4, 710; M. Meldal, in Neoglycoconjugates: Preparation and Applications, supra; S. J. Danishefsky; J. Y. Roberge, in Glycopeptides and Related Compounds: Chemical Synthesis, Analysis and Applications, 1995, D. G. Large, C. D. Warren, Eds., Marcel Dekker, New York; S. T. Cohen-Anisfeld and P. T. Lansbury, Jr., J. Am. Chem. Soc., 1993, 115, 10531; S. T. Anisfeld; P. T. Lansbury jr., J. Org. Chem, 1990, 55, 5560; D. Vetter, et al., Angew. Chem. Int. Ed. Engl, 1995, 34, 60-63). Cohen-Anisfeld and Lansbury disclosed a convergent solution-based coupling of selected already available saccharides with peptides. S. T. Cohen-Anisfeld; P. T. Lansbury, Jr., J. Am. Chem. Soc., supra.
Thus, few effective methods for the preparation of xcex1O-linked glycoconjugates were known prior to the present invention. Nakahara, Y., et al., In Synthetic Oligosaccharides, ACS Symp. Ser. 560, 1994, pp. 249-266; Garg, H. G., et al., Adv. Carb. Chem. Biochem. 1994, 50, 277. Nearly all approaches incorporated the amino acid (serine or threonine) at the monosaccharide stage. This construction would be followed by elaboration of the peptidyl and carbohydrate domains in a piecemeal fashion. Qui, D.; Koganty, R. R.; Tetrahedron Lett. 1997, 38, 45. Eloffson, M., et al., Tetrahedron 1997, 53, 369. Meinjohanns, E., et al., J. Chem. Soc., Perkin Trans. 1, 1996, 985. Wang, Z -G., et al., Carbohydr. Res. 1996, 295, 25. Szabo, L., et al., Carbohydr. Res. 1995, 274, 11. The scope of the synthetic problem is well known in the art, but little progress has been achieved. The present invention provides an alternate, simpler and more convergent approach (FIG. 2).
Toyokuni et al., J. Amer. Chem. Soc., 1994, 116, 395, have prepared synthetic vaccines comprising dimeric Tn antigen-lipopeptide conjugates having efficacy in eliciting an immune response against Tn-expressing glycoproteins. However, prior to investigations of the present inventors, it was not appreciated that the surface of prostate cancer cells presents glycoproteins comprising Tn clusters linked via threonine rather than serine residues. Accordingly, the present invention provides a vaccine having unexpectedly enhanced anticancer efficacy.
Accordingly, one object of the present invention is to provide novel xcex1-O-linked glycoconjugates including glycopeptides and related compounds which are useful as anticancer therapeutics.
Another object of the present invention is to provide synthetic methods for preparing such glycoconjugates. An additional object of the invention is to provide compositions useful in the treatment of subjects suffering from cancer comprising any of the glycoconjugates available through the preparative methods of the invention, optionally in combination with pharmaceutical carriers.
The present invention is also intended to provide a fully synthetic carbohydrate vaccine capable of fostering active immunity in humans.
A further object of the invention is to provide methods of treating subjects suffering from of cancer using any of the glycoconjugates available through the preparative methods of the invention, optionally in combination with pharmaceutical carriers.